The overall goal of this project is to develop a new paradigm for targeting the HIV-1 envelope in order to inactivate the virus specifically, and before host cell encounter. The uniquely HIV-1-specific envelope spike protein, comprised of the trimeric complex of gp120 and gp41 heterodimers embedded in the virus membrane, is the only virus-specific protein exposed on the outside of the virion particle and thus is an attractive targt for neutralizing the virus. HIV-1 inactivation by targeting the Env protein could prevent initial infection, and suppress virus spread in already-infected individuals. Recently, we reported the discovery of a molecule termed CVN-DAVEI that selectively induced destructive lysis of both fully infectious BaL HIV-1 and pseudotyped virus. Our vision is that CVN-DAVEI takes advantage of the inherent metastability of the Env complex to destroy the virus, and that this metastability is an Achilles' heel against which a new class of HIV- inactivating hunter-killer molecules can be designed. While the HIV-1 inactivation phenotype is enticing, our understanding of the structural mechanism of DAVEI function is incomplete. In this proposal, we will determine this mechanism and use the resulting mechanistic underpinning to move towards smaller and more clinically relevant HIV-1 inactivator lead compounds. Our project team will embody the required protein science, molecular design, chemical synthesis and computational structure analysis expertise to accomplish these overarching goals. Three interconnected Specific Aims are proposed. Aim 1. Determine the mechanism of the DAVEI MPER domain in the lytic inactivation of HIV-1. Aim 2. Determine the mechanism of Env glycan engagement by the DAVEI lectin domain and the stoichiometry of DAVEI:Env spike encounter. Aim 3. Define the roles of Env structure and conformational plasticity in the lytic inactivation of HIV-1 by DAVEI via the design of novel DAVEI fusion constructs. Overall, this work will define the minimum requirements for virolytic function by dual-binding molecules. In turn, the results will expand our understanding on how the virus metastability, that is a requirement for HIV-1 cell infection, can be exploited for DAVEI-induced HIV-1 lysis. The project will yield hunter-killer lead compounds that will open up new molecular design strategies for preventing transmission and battling infection. Finally, the virolysis activity engineered into these anti-HIV constructs wll provide precedent to design molecules to combat other metastable enveloped viruses, such as influenza, Ebola, and dengue.